Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi-centre setting.

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection. Here we present the validation experiments of the assay in several large AML research centres, both in Europe and the United States. Variability within instruments and sample processing showed high correlations between different instruments (Rpearson  > 0·91, P < 0·001). Multi-centre testing introduced variation in reported LSC percentages but was found to be below the clinical relevant threshold. Clear gating protocols resulted in all laboratories being able to perform LSC assessment of the validation set. Participating centres were nearly unanimously able to distinguish LSChigh (>0·03% LSC) from LSClow (<0·03% LSC) despite inter-laboratory variation in reported LSC percentages. This study proves that the LSC assay is highly reproducible. These results together with the high prognostic impact of LSC load at diagnosis in AML patients render the one-tube LSC assessment a good marker for future risk classification.

Absence of leukaemic CD34+ cells in acute myeloid leukaemia is of high prognostic value: a longstanding controversy deciphered.

Primary resistance and relapses after initial successful treatment are common in acute myeloid leukaemia and therefore outcome remains poor. More accurate risk group stratification and effective personalized risk adapted treatment are necessary to improve outcome. In the last two decades, controversial results have been published concerning the prognostic relevance of CD34 expression. In this study of 706 acute myeloid leukaemia patients, we established a new flow cytometric-based CD34-definition, without use of cut-off values. We discriminated CD34-positive (n = 548) and CD34-negative (n = 158) patients by the presence or absence of neoplastic CD34+ cells, respectively. CD34-status was defined using aberrant immunophenotypes and validated using molecular phenotypes. This new definition of CD34 enables strong prediction of treatment outcome in the entire patient group and in several risk subgroups. Previously observed discrepancies in prognostic impact of CD34 protein expression using cut-offs (5-20%) can now entirely be explained by considering the number of CD34-negative cases. In the total patient group, the absence of neoplastic CD34-positive cells is paralleled by low levels of minimal residual disease, suggesting relative therapy sensitivity and explaining longer survival. Overall, we present CD34 surface expression as a relatively simple, powerful and independent predictor of clinical outcome, now warranting incorporation in acute myeloid leukaemia risk stratification.

A Phase 2 study of bortezomib combined with either idarubicin/cytarabine or cytarabine/etoposide in children with relapsed, refractory or secondary acute myeloid leukemia: a report from the Children's Oncology Group.

BACKGROUND: This Phase 2 study tested the tolerability and efficacy of bortezomib combined with reinduction chemotherapy for pediatric patients with relapsed, refractory or secondary acute myeloid leukemia (AML). Correlative studies measured putative AML leukemia initiating cells (AML-LIC) before and after treatment. PROCEDURE: Patients with <400 mg/m(2) prior anthracycline received bortezomib combined with idarubicin (12 mg/m(2) days 1-3) and low-dose cytarabine (100 mg/m(2) days 1-7) (Arm A). Patients with ≥400 mg/m(2) prior anthracycline received bortezomib with etoposide (100 mg/m(2) on days 1-5) and high-dose cytarabine (1 g/m(2) every 12 hours for 10 doses) (Arm B). RESULTS: Forty-six patients were treated with 58 bortezomib-containing cycles. The dose finding phase of Arm B established the recommended Phase 2 dose of bortezomib at 1.3 mg/m(2) on days 1, 4, and 8 with Arm B chemotherapy. Both arms were closed after failure to meet predetermined efficacy thresholds during the first stage of the two-stage design. The complete response (CR + CRp) rates were 29% for Arm A and 43% for Arm B. Counting additional CRi responses (CR with incomplete neutrophil recovery), the overall CR rates were 57% for Arm A and 48% for Arm B. The 2-year overall survival (OS) was 39 ± 15%. Correlative studies showed that LIC depletion after the first cycle was associated with clinical response. CONCLUSION: Bortezomib is tolerable when added to chemotherapy regimens for relapsed pediatric AML, but the regimens did not exceed preset minimum response criteria to allow continued accrual. This study also suggests that AML-LIC depletion has prognostic value.

Impact of hemodilution on flow cytometry based measurable residual disease assessment in acute myeloid leukemia.

Measurable residual disease (MRD) measured in the bone marrow (BM) of acute myeloid leukemia (AML) patients after induction chemotherapy is an established prognostic factor. Hemodilution, stemming from peripheral blood (PB) mixing within BM during aspiration, can yield false-negative MRD results. We prospectively examined hemodilution by measuring MRD in BM aspirates obtained from three consecutive 2 mL pulls, along with PB samples. Our results demonstrated a significant decrease in MRD percentages between the first and second pulls (P = 0.025) and between the second and third pulls (P = 0.025), highlighting the impact of hemodilution. Initially, 39% of MRD levels (18/46 leukemia-associated immunophenotypes) exceeded the 0.1% cut-off, decreasing to 30% (14/46) in the third pull. Additionally, we assessed the performance of six published methods and parameters for distinguishing BM from PB samples, addressing or compensating for hemodilution. The most promising results relied on the percentages of CD16dim granulocytic population (scarce in BM) and CD117high mast cells (exclusive to BM). Our findings highlight the importance of estimating hemodilution in MRD assessment to qualify MRD results, particularly near the common 0.1% cut-off. To avoid false-negative results by hemodilution, it is essential to collect high-quality BM aspirations and preferably utilizing the initial pull for MRD testing.

Analytical assay validation for acute myeloid leukemia measurable residual disease assessment by multiparametric flow cytometry.

BACKGROUND: Measurable residual disease (MRD) assessed by multiparametric flow cytometry (MFC) has gained importance in clinical decision-making for acute myeloid leukemia (AML) patients. However, complying with the recent In Vitro Diagnostic Regulations (IVDR) in Europe and Food and Drug Administration (FDA) guidance in the United States requires rigorous validation prior to their use in investigational clinical trials and diagnostics. Validating AML MRD-MFC assays poses challenges due to the unique underlying disease biology and paucity of patient specimens. In this study, we describe an experimental framework for validation that meets regulatory expectations. METHODS: Our validation efforts focused on evaluating assay accuracy, analytical specificity, analytical and functional sensitivity (limit of blank (LoB), detection (LLoD) and quantitation (LLoQ)), precision, linearity, sample/reagent stability and establishing the assay background frequencies. RESULTS: Correlation between different MFC methods was highly significant (r = 0.99 for %blasts and r = 0.93 for %LAIPs). The analysis of LAIP specificity accurately discriminated from negative control cells. The assay demonstrated a LoB of 0.03, LLoD of 0.04, and LLoQ of 0.1%. Precision experiments yielded highly reproducible results (Coefficient of Variation <20%). Stability experiments demonstrated reliable measurement of samples up to 96 h from collection. Furthermore, the reference range of LAIP frequencies in non-AML patients was below 0.1%, ranging from 0.0% to 0.04%. CONCLUSION: In this manuscript, we present the validation of an AML MFC-MRD assay using BM/PB patient specimens, adhering to best practices. Our approach is expected to assist other laboratories in expediting their validation activities to fulfill recent health authority guidelines.

AML/Normal Progenitor Balance Instead of Total Tumor Load (MRD) Accounts for Prognostic Impact of Flowcytometric Residual Disease in AML.

Measurable residual disease (MRD) in AML, assessed by multicolor flow cytometry, is an important prognostic factor. Progenitors are key populations in defining MRD, and cases of MRD involving these progenitors are calculated as percentage of WBC and referred to as white blood cell MRD (WBC-MRD). Two main compartments of WBC-MRD can be defined: (1) the AML part of the total primitive/progenitor (CD34+, CD117+, CD133+) compartment (referred to as primitive marker MRD; PM-MRD) and (2) the total progenitor compartment (% of WBC, referred to as PM%), which is the main quantitative determinant of WBC-MRD. Both are related as follows: WBC-MRD = PM-MRD × PM%. We explored the relative contribution of each parameter to the prognostic impact. In the HOVON/SAKK study H102 (300 patients), based on two objectively assessed cut-off points (2.34% and 10%), PM-MRD was found to offer an independent prognostic parameter that was able to identify three patient groups with different prognoses with larger discriminative power than WBC-MRD. In line with this, the PM% parameter itself showed no prognostic impact, implying that the prognostic impact of WBC-MRD results from the PM-MRD parameter it contains. Moreover, the presence of the PM% parameter in WBC-MRD may cause WBC-MRD false positivity and WBC-MRD false negativity. For the latter, at present, it is clinically relevant that PM-MRD ≥ 10% identifies a patient sub-group with a poor prognosis that is currently classified as good prognosis MRDnegative using the European LeukemiaNet 0.1% consensus MRD cut-off value. These observations suggest that residual disease analysis using PM-MRD should be conducted.

MRD Tailored Therapy in AML: What We Have Learned So Far.

Acute myeloid leukemia (AML) is a heterogeneous clonal disease associated with a dismal survival, partly due to the frequent occurrence of relapse. Many patient- and leukemia-specific characteristics, such as age, cytogenetics, mutations, and measurable residual disease (MRD) after intensive chemotherapy, have shown to be valuable prognostic factors. MRD has become a rich field of research where many advances have been made regarding technical, biological, and clinical aspects, which will be the topic of this review. Since many laboratories involved in AML diagnostics have experience in immunophenotyping, multiparameter flow cytometry (MFC) based MRD is currently the most commonly used method. Although molecular, quantitative PCR based techniques may be more sensitive, their disadvantage is that they can only be applied in a subset of patients harboring the genetic aberration. Next-generation sequencing can assess and quantify mutations in many genes but currently does not offer highly sensitive MRD measurements on a routine basis. In order to provide reliable MRD results, MRD assay optimization and standardization is essential. Different techniques for MRD assessment are being evaluated, and combinations of the methods have shown promising results for improving its prognostic value. In this regard, the load of leukemic stem cells (LSC) has also been shown to add to the prognostic value of MFC-MRD. At this moment, MRD after intensive chemotherapy is most often used as a prognostic factor to help stratify patients, but also to select the most appropriate consolidation therapy. For example, to guide post-remission treatment for intermediate-risk patients where MRD positive patients receive allogeneic stem cell transplantation and MRD negative receive autologous stem cell transplantation. Other upcoming uses of MRD that are being investigated include: selecting the type of allogeneic stem cell transplantation therapy (donor, conditioning), monitoring after stem cell transplantation (to allow intervention), and determining drug efficacy for the use of a surrogate endpoint in clinical trials.

Activin-A Induces Fewer, but Larger Osteoclasts From Monocytes in Both Healthy Controls and Fibrodysplasia Ossificans Progressiva Patients.

Fibrodysplasia Ossificans Progressiva (FOP) is a rare genetic disease characterized by heterotopic ossification (HO) that occurs in muscle tissue, tendons, and ligaments. The disease is caused by mutations in the Activin receptor type I (ACVR1) gene resulting in enhanced responsiveness to Activin-A. Binding of this molecule to the mutated receptor induces HO. Bone metabolism normally requires the coupled action of osteoblasts and osteoclasts, which seems to be disturbed during HO. We hypothesize that Activin-A may also counteract the formation of osteoclasts in FOP patients. In this study we investigated the effect of Activin-A on osteoclast differentiation of CD14+ monocytes from FOP patients and healthy controls. The lymphocytic and monocytic cell populations were determined by FACS analysis. Expression of the mutated R206H receptor was assessed and confirmed by allele specific PCR. The effect of Activin-A on osteoclastogenesis was assessed by counting the number and size of multinucleated cells. Osteoclast activity was determined by culturing the cells on Osteo Assay plates. The influence of Activin-A on expression of various osteoclast related genes was studied with QPCR. Blood from FOP patients contained similar percentages of classical, intermediate, or non-classical monocytes as healthy controls. Addition of Activin-A to the osteoclastogenesis cultures resulted in fewer osteoclasts in both control and FOP cultures. The osteoclasts formed in the presence of Activin-A were, however, much larger and more active compared to the cultures without Activin-A. This effect was tempered when the Activin-A inhibitor follistatin was added to the Activin-A containing cultures. Expression of osteoclast specific genes Cathepsin K and TRAcP was upregulated, gene expression of osteoclastogenesis related genes M-CSF and DC-STAMP was downregulated by Activin-A. Since Activin-A is a promising target for inhibiting the formation of HO in FOP, it is important to know its effects on both osteoblasts and osteoclasts. Our study shows that Activin-A induces fewer, but larger and more active osteoclasts independent of the presence of the mutated ACVR1 receptor. When considering FOP as an Activin-A driven disease that acts locally, our findings suggest that Activin-A could cause a more pronounced local resorption by larger osteoclasts. Thus, when targeting Activin-A in patients with neutralizing antibodies, HO formation could potentially be inhibited, and osteoclastic activity could be slightly reduced, but then performed dispersedly by more and smaller osteoclasts.

Identification of a small subpopulation of candidate leukemia-initiating cells in the side population of patients with acute myeloid leukemia.

In acute myeloid leukemia (AML), apart from the CD34(+)CD38(-) compartment, the side population (SP) compartment contains leukemic stem cells (LSCs). We have previously shown that CD34(+)CD38(-) LSCs can be identified using stem cell-associated cell surface markers, including C-type lectin-like molecule-1 (CLL-1), and lineage markers, such as CD7, CD19, and CD56. A similar study was performed for AML SP to further characterize the SP cells with the aim of narrowing down the putatively very low stem cell fraction. Fluorescence-activated cell sorting (FACS) analysis of 48 bone marrow and peripheral blood samples at diagnosis showed SP cells in 41 of 48 cases that were partly or completely positive for the markers, including CD123. SP cells in normal bone marrow (NBM) were completely negative for markers, except CD123. Further analysis revealed that the SP fraction contains different subpopulations: (a) three small lymphoid subpopulations (with T-, B-, or natural killer-cell markers); (b) a differentiated myeloid population with high forward scatter (FSC(high)) and high sideward scatter (SSC(high)), high CD38 expression, and usually with aberrant marker expression; (c) a more primitive FSC(low)/SSC(low), CD38(low), marker-negative myeloid fraction; and (d) a more primitive FSC(low)/SSC(low), CD38(low), marker-positive myeloid fraction. NBM contained the first three populations, although the aberrant markers were absent in the second population. Suspension culture assay showed that FSC(low)/SSC(low) SP cells were highly enriched for primitive cells. Fluorescence in situ hybridization (FISH) analyses showed that cytogenetically abnormal colonies originated from sorted marker positive cells, whereas the cytogenetically normal colonies originated from sorted marker-negative cells. In conclusion, AML SP cells could be discriminated from normal SP cells at diagnosis on the basis of expression of CLL-1 and lineage markers. This reveals the presence of a low-frequency (median, 0.0016%) SP subfraction as a likely candidate to be enriched for leukemia stem cells.

Correlation of minimal residual disease cell frequency with molecular genotype in patients with acute myeloid leukemia.

BACKGROUND: About 70-80 percent of patients with acute myeloid leukemia enter complete remission, but at least half of these patients who achieve remission go on to relapse. Improved treatment is likely to come from increasing the time to relapse, especially for younger patients. With the vastly increasing number of targeted therapies there is a strong need for short-term end-points to efficiently test such therapies for further pursuance. Minimal residual disease assessment may offer such an end-point since it is a strong independent prognostic factor. As proof of principle we examined this concept for FLT3-ITD status at diagnosis. DESIGN AND METHODS: We determined FLT3-ITD status in bone marrow samples from 196 patients with newly diagnosed acute myeloid leukemia. The frequencies of residual leukemic cells of these 196 patients were assessed in 267 follow-up bone marrow samples using immunophenotypic assessment of minimal residual disease. RESULTS: The median frequency of residual leukemic cells after the first cycle of chemotherapy was 8.5-fold higher in patients with FLT3-ITD than in those with wild type FLT3. Such a difference translates into differences in survival, even if other potentially outcome-modulating mutations, such as NPM1, KIT, NRAS, KRAS, FLT3-exon 20 and PTPN11 are included in the analysis. CONCLUSIONS: This study shows that it could be possible to study the efficacy of FLT3 inhibitors using the level of minimal residual disease as a short-term end-point.

Immunophenotypic Detection of Measurable Residual (Stem Cell) Disease Using LAIP Approach in Acute Myeloid Leukemia.

Half of the patients with acute myeloid leukemia (AML), who achieve complete remission after chemotherapy treatment, will ultimately experience a relapse. Measurable residual disease (MRD) is an important post-treatment risk factor in AML, because it gives additional information about the depth of the remission. Within MRD, the small population of leukemic stem cells (LSCs) is thought to be at the base of the actual relapse. In this protocol, the flow cytometric detection of MRD and LSCs herein is outlined. We give a detailed overview of the sampling procedures for optimal multiparameter flow cytometry assessment of both MRD and LSC, using leukemia associated immunophenotypes (LAIPs) and LSC markers. Moreover, an overview of the gating strategies to detect LAIPs and LSC markers is provided. This protocol serves as guidance for flow cytometric detection of measurable residual (stem cell) disease necessary for proper therapeutic decision making in AML patients. © 2019 The Authors. Basic Protocol 1: Immunophenotypic LAIP detection for measurable residual disease monitoring Basic Protocol 2: Immunophenotypic detection of CD34+CD38- leukemic stem cells.

Relationship between CD34/CD38 and side population (SP) defined leukemia stem cell compartments in acute myeloid leukemia.

Leukemic stem cells (LSCs), defined by CD34/CD38 expression, are believed to be essential for leukemia initiation and therapy resistance in acute myeloid leukemia. In addition, the side population (SP), characterized by high Hoechst 33342 efflux, reflecting therapy resistance, has leukemia initiating ability. The purpose of this study is, in both CD34-positive and CD34-negative AML, to integrate both types of LSC compartment into a new more restricted definition. Different CD34/CD38/SP defined putative LSC and normal hematopoietic compartments, with neoplastic or normal nature, respectively, were thus identified after cell sorting, and confirmed by FISH/PCR. Stem cell activity was assessed in the long-term liquid culture stem cell assay. SP fractions harbored the strongest functional stem cell activity in both normal and neoplastic cells in both CD34-positive and CD34-negative AML. Overall, inclusion of SP fraction decreased the size of the putative CD34/CD38 defined LSC compartment by a factor >500. For example, for the important CD34+CD38- LSC compartment, the median SP/CD34+CD38- frequency was 5.1 per million WBC (CD34-positive AML), and median SP/CD34-CD38+ frequency (CD34-negative AML) was 1796 per million WBC. Improved detection of LSC may enable identification of therapy resistant clones, and thereby identification of novel LSC specific, HSC sparing, therapies.

Immunophenotypic measurable residual disease (MRD) in acute myeloid leukemia: Is multicentric MRD assessment feasible?

Flow-cytometric detection of now termed measurable residual disease (MRD) in acute myeloid leukemia (AML) has proven to have an independent prognostic impact. In a previous multicenter study we developed protocols to accurately define leukemia-associated immunophenotypes (LAIPs) at diagnosis. It has, however, not been demonstrated whether the use of the defined LAIPs in the same multicenter setting results in a high concordance between centers in MRD assessment. In the present paper we evaluated whether interpretation of list-mode data (LMD) files, obtained from MRD assessment of previously determined LAIPs during and after treatment, could reliably be performed in a multicenter setting. The percentage of MRD positive cells was simultaneously determined in totally 173 LMD files from 77 AML patients by six participating centers. The quantitative concordance between the six participating centers was meanly 84%, with slight variation of 75%-89%. In addition our data showed that the type and number of LAIPs were of influence on the performance outcome. The highest concordance was observed for LAIPs with cross-lineage expression, followed by LAIPs with an asynchronous antigen expression. Our results imply that immunophenotypic MRD assessment in AML will only be feasible when fully standardized methods are used for reliable multicenter assessment.

CD34+CD38- leukemic stem cell frequency to predict outcome in acute myeloid leukemia.

Current risk algorithms are primarily based on pre-treatment factors and imperfectly predict outcome in acute myeloid leukemia (AML). We introduce and validate a post-treatment approach of leukemic stem cell (LSC) assessment for prediction of outcome. LSC containing CD34+CD38- fractions were measured using flow cytometry in an add-on study of the HOVON102/SAKK trial. Predefined cut-off levels were prospectively evaluated to assess CD34+CD38-LSC levels at diagnosis (n = 594), and, to identify LSClow/LSChigh (n = 302) and MRDlow/MRDhigh patients (n = 305) in bone marrow in morphological complete remission (CR). In 242 CR patients combined MRD and LSC results were available. At diagnosis the CD34+CD38- LSC frequency independently predicts overall survival (OS). After achieving CR, combining LSC and MRD showed reduced survival in MRDhigh/LSChigh patients (hazard ratio [HR] 3.62 for OS and 5.89 for cumulative incidence of relapse [CIR]) compared to MRDlow/LSChigh, MRDhigh/LSClow, and especially MRDlow/LSClow patients. Moreover, in the NPM1mutant positive sub-group, prognostic value of golden standard NPM1-MRD by qPCR can be improved by addition of flow cytometric approaches. This is the first prospective study demonstrating that LSC strongly improves prognostic impact of MRD detection, identifying a patient subgroup with an almost 100% treatment failure probability, warranting consideration of LSC measurement incorporation in future AML risk schemes.

Formation of osteoclast-like cells from peripheral blood of periodontitis patients occurs without supplementation of macrophage colony-stimulating factor.

AIM: To determine whether peripheral blood mononuclear cells (PBMCs) from chronic periodontitis patients differ from PBMCs from matched control patients in their capacity to form osteoclast-like cells. MATERIAL AND METHODS: PBMCs from 10 subjects with severe chronic periodontitis and their matched controls were cultured on plastic or on bone slices without or with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL). The number of tartrate-resistant acid phosphatase-positive (TRACP(+)) multinucleated cells (MNCs) and bone resorption were assessed. RESULTS: TRACP(+) MNCs were formed under all culture conditions, in patient and control cultures. In periodontitis patients, the formation of TRACP(+) MNC was similar for all three culture conditions; thus supplementation of the cytokines was not needed to induce MNC formation. In control cultures, however, M-CSF or M-CSF/RANKL resulted in higher numbers compared with cultures without cytokines. Upregulations of osteoclast marker mRNA cathepsin K and carbonic anhydrase II confirmed the osteoclastic character. Bone resorption was only observed when PBMCs were cultured in the presence of M-CSF and RANKL. CONCLUSION: Our data indicate that PBMCs from periodontitis patients do not need priming by M-CSF to become osteoclast-like cells, suggesting that PBMCs from periodontitis patients are present in the circulation in a different state of activity.

Measurable residual disease in acute myeloid leukemia using flow cytometry: approaches for harmonization/standardization.

Introduction: Measurable residual disease (MRD) in acute myeloid leukemia (AML) is a rapidly evolving area with many institutes embarking on it, both in academic and pharmaceutical settings. However, there is a multitude of approaches to design, perform, and report flow cytometric MRD. Together with the long-term experience needed, this makes flow cytometric MRD in AML nonstandardized and time-consuming. Areas covered: This paper briefly summarizes critical issues, like sample preparation and transport, markers and fluorochromes of choice, but in particular focuses on the main issues, which includes specificity and sensitivity, hereby providing a new model that may circumvent the main disadvantages of the present approaches. New approaches that may add to the value of flow cytometric MRD includes assessment of leukemia stem cells, MRD in peripheral blood, and approaches to use multidimensional image analysis. Expert commentary: MRD in AML requires standardization/harmonization on many aspects, for which the present paper offers possible guidelines.

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.